Alteration of the daunorubicin-triggered sphingomyelin-ceramide pathway and apoptosis in MDR cells: influence of drug transport abnormalities.

We have previously shown that in myeloid leukemic cells, daunorubicin (DNR) induces apoptosis via the activation of the sphingomyelin-ceramide pathway. We have now investigated sphingomyelin (SM) hydrolysis, ceramide generation, and apoptosis in vincristine-selected multidrug resistant (MDR) HL-60 cells (HL-60/Vinc), compared with their parental counterparts. We show that DNR triggers the SM cycle (stimulation of neutral sphingomyelinase, SM hydrolysis, and ceramide generation) and apoptosis in both parental and MDR cells, when used at isotoxic doses (ie., 1 and 100 microM for HL-60 and HL-60/Vinc, respectively). However, in MDR cells treated with either 10 microM DNR or 1 microM DNR in association with the P-glycoprotein (P-gp) blocker verapamil (treatment conditions which yield an intracellular DNR concentration similar to that achieved with 1 microM in the parental cells), we were unable to detect SM hydrolysis, ceramide generation and apoptosis. This implies that inhibition of the DNR-induced SM cycle in MDR cells is not directly related to P-gp. We have also investigated the influence of intracellular drug localization on the DNR-induced SM-cycle in HL-60/Vinc cells. In these cells, DNR at 10 microM is mainly localized in cytoplasmic vesicles, while the drug is diffusely distributed when used at 100 microM. A diffuse distribution pattern was also observed when MDR cells were treated with 1 microM DNR in association with the cyclosporine derivative PSC-833, but not with verapamil. In parallel, PSC-833, but not verapamil, restored the induction of the SM cycle and the apoptotic potential of DNR, and markedly increased drug cytotoxicity in MDR cells. Our results suggest that altered intracellular drug transport plays an important role in limiting ceramide generation and cell death in MDR cells.

Dual mechanism of daunorubicin-induced cell death in both sensitive and MDR-resistant HL-60 cells.

Exposure of some acute myeloid leukaemia (AML) cells to daunorubicin leads to rapid cell death, whereas other AML cells show natural drug resistance. This has been attributed to expression of functional P-glycoprotein resulting in reduced drug accumulation. However, it has also been proposed that P-glycoprotein-expressing multidrug-resistant (MDR) cells are inherently defective for apoptosis. To distinguish between these different possibilities, we have compared the cell death process in a human AML cell line (HL-60) with a MDR subline (HL-60/Vinc) at doses that yield either similar intracellular daunorubicin concentrations or comparable cytotoxicity. Adjustment of the dose to obtain the same intracellular drug accumulation in the two cell lines did not result in equal cytotoxicity, suggesting the presence of additional resistance mechanisms in the P-glycoprotein-expressing HL-60/Vinc cells. However, at equitoxic doses, similar cell death pathways were observed. In HL-60 cells, daunorubicin induced rapid apoptosis at 0.5-1 microM and delayed mitotic cell death at 0.1 microM. These concentrations are within the clinical dose range. Similarly, HL-60/Vinc cells underwent apoptosis at 50-100 microM daunorubicin and mitotic cell death at 10 microM. These results show, for the first time, that anthracyclines can induce cell death by a dual mechanism in both sensitive and MDR cells. Our results also show that not only the cytotoxicity, but also the kinetics and mechanism of cell death, are dose dependent. Interestingly, regrowth was observed only in association with delayed cell death and the formation of enlarged, often polyploid, cells with micronucleation, suggesting that morphological criteria may be useful to evaluate treatment efficacy in patients with myeloid leukaemias.

Opposite effects of tumor necrosis factor alpha on the sphingomyelin-ceramide pathway in two myeloid leukemia cell lines: role of transverse sphingomyelin distribution in the plasma membrane.

Tumor necrosis factor alpha (TNF alpha) mediates proliferation, functional activation, and apoptotic cell death depending on the target cell type. Although sphingomyelin (SPM) hydrolysis and ceramide generation may function as an important mediator in TNF alpha signaling, the molecular mechanisms of the signaling pathway(s) are still not well understood. The aim of the present study is to compare the effect of TNF alpha on SPM metabolism and cell growth in two myeloid leukemic cell lines (U937 and KG1a) that differ in their sensitivity to TNF alpha. Our results show that TNF alpha induced apoptosis in U937 but not in KG1a cells. TNF alpha triggered in KG1a cells neither SPM hydrolysis nor ceramide generation, but induced SPM synthesis and ceramide breakdown as well as dose-dependent cell proliferation. In contrast, TNF alpha induced in U937 SPM hydrolysis and ceramide generation as well as dose-dependent cell death. Synthetic cell permeant ceramide, as well as natural ceramide, generated by treatment with bacterial sphingomyelinase (SPMase), all induced apoptosis in both U937 and KG1a cells. These findings indicate that the SPM-ceramide pathway is altered in KG1a cells upstream of the ceramide generation. Analysis of the transverse distribution of SPM in the plasma membrane showed that the SPM pool involved in cell signaling (inner leaflet) was markedly reduced in KG1a cells; it is 7-fold lower than that found in the inner leaflet of U937 cells. Therefore, our study strongly suggests that the different responses induced by TNF alpha in myeloid cells are dependent on the SPM plasma membrane transverse asymmetry.

Daunorubicin-induced internucleosomal DNA fragmentation in acute myeloid cell lines.

The study was designed to evaluate the implication of apoptosis in myeloid leukemic cell death induced by daunorubicin (DNR) and to identify the possible factors which may influence this process. DNR-induced apoptosis was characterized by morphology and DNA fragmentation in six leukemic myeloid cell lines which expressed different differentiation phenotypes. In phenotypically mature HL-60 and U937 cells, DNR induced typical apoptosis with characteristic morphological changes and intense internucleosomal DNA fragmentation within a narrow concentration range (0.5-2 microM). When these cells were treated with higher doses of DNR, large DNA fragments (100 kbp), but not internucleosomal fragments, were identified. DNR-induced DNA fragmentation in HL-60 and U937 was inhibited by antioxidants such as N-acetylcysteine (N-ac) or pyrrolidine-dithiocarbamate (PDTC). In the phenotypically immature KG1a, KG1, HEL and ML1 cell lines DNR induced no characteristic apoptotic morphological features as well as very low levels of internucleosomal DNA fragmentation, whereas large DNA fragments (200 kbp) were observed in KG1a treated with 7 microM DNR. Since the latter expressed P-glycoprotein (P-gp), the role of P-gp in the lack of apoptotic response to DNR was investigated. One P-gp inhibitor (verapamil) slightly improved DNR-induced DNA fragmentation in KG1a cells whereas the combination of verapamil and buthionine-sulfoximine (BSO), which depletes glutathion store, further increased internucleosomal DNA fragmentation. In conclusion, DNR induced internucleosomal DNA fragmentation in some but not all AML cells; the magnitude of this process being influenced by both intracellular drug concentration and oxidative balance.

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.